Project Summary/Abstract Inflammatory Bowel Disease (IBD), is increasingly linked with disruption of the normal gut microbial community. While many studies correlate disease with increases or decreases of certain microbes, the role of these microbes in IBD is currently unknown. Microbes largely sense and respond to their environment through metabolites and small molecules. This study?s premise is that members of the gut microbiome produce molecules which exacerbate or diminish inflammatory signals, thereby progressing or retarding inflammation and tissue damage in patients. In preliminary studies, patient-derived bacteria were grown and separated into fractions which were screened for molecules that promote inflammation. This effort identified several initial hits, one being a lipidic molecule from Bifidobacterium breve. Projects proposed in Aim 1 of this grant will identify and characterize this molecule and its mechanism of action. The success of this preliminary work prompted us to propose a screen of an entire library of gut microbial fractions to identify potent immunomodulatory molecules in Aim 2. To this end, three screens will be performed on: 1) wild-type, 2) TLR2-/- TLR4-/-, and 3) LPS-stimulated mouse dendritic cells. While the wild-type cells provide a broad view of all inflammatory antigens, the TLR2-/- TLR4-/- cells, which lack the common receptors TLR2 and TLR4, reveal only molecules that act through less- studied pathways. The LPS-stimulation assay will identify anti-inflammatory molecules (i.e. molecules that inhibit the LPS response). In Aim 3, hit fractions from all assays will be confirmed and characterized structurally and functionally via an array of analytical techniques. Molecules that are highly potent, purified, and with a solved structure, will undergo secondary assays to probe their mechanism of action. Initial secondary assays will include ELISAs with deletion cell lines (lacking specific immune receptors) to identify which signaling pathways are responsible for the bioactivity. This research will be performed primarily at Harvard Medical School which houses state of the art facilities and equipment for performing chemical analysis (e.g. NMR, HPLC, MS). Some aspects of this work will also be performed at the Broad Institute which provides access to bacterial isolates, mouse strains, as well as an array of equipment and resources for immunological and secondary assays. The training plan for the applicant will include frequent communication with mentors about the project progress and career plans. Pertinent courses, workshops, and conferences will be attended by the applicant to develop networking and presentation skills, as well as help in rigorous assessment of the science in this proposal. 1